CN101576653A - Method for washing and recycling membranate glass slide for microdissection - Google Patents
Method for washing and recycling membranate glass slide for microdissection Download PDFInfo
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- CN101576653A CN101576653A CNA2009100121560A CN200910012156A CN101576653A CN 101576653 A CN101576653 A CN 101576653A CN A2009100121560 A CNA2009100121560 A CN A2009100121560A CN 200910012156 A CN200910012156 A CN 200910012156A CN 101576653 A CN101576653 A CN 101576653A
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Abstract
The invention discloses technology for washing and recycling a membranate glass slide for microdissection. The technology can be implemented according to the following steps: (1) dipping and shaking the membranate glass slide in mixed solution A of boric acid, alcohol and EDTA; (2) washing the membranate by using double distilled water, and transferring the membranate glass slide into prolease K solution to incubate; (3) placing the glass slide into clean water; slightly touching a sample on the membrane by a finger; and washing the membranate glass slide by the double distilled water; (4) dipping the glass slide in mixed solution B of hydrochloric acid, the alcohol and the EDTA; washing the membranate glass slide by using the double distilled water and absolute ethyl alcohol; and finishing the last washing in the absolute ethyl alcohol; and (5) quickly transferring the membranate glass slide onto a super-clean bench; quickly drying the absolute ethyl alcohol; and carrying out ultraviolet radiation at the same time. The technology removes tissues, nucleic acid, albumen and other micromolecular substances remained on the surface of a consumptive material, and can sufficiently maintain the completeness and adhering effect of the membranate glass slide for microdissection.
Description
Technical field
The present invention relates to micro-dissections molecular biology section cleaning field, a kind of specifically cleaning reutilization technology of micro-dissections film microslide.
Background technology
The appearance of laser capture micro-dissections (Laser Capture Microdissection) technology and system thereof, make the related scientific research worker can accurately separate purpose tissue specimen to be studied, cell (living cells of in vitro culture and be fixed on histocyte in the section), organelle even chromosomal region band, be used for follow-up study, the accuracy of its control sample is subjected to the favor of whole world researcher, and at present most strong research institutions all have been equipped with this technology and related facility.
When carrying out micro-dissections with histotomy, need use a kind of film microslide consumptive material of special use, it costs an arm and a leg, and simultaneously, because the purpose sample that is used to study needs the enough quantity of enrichment, makes that this consumptive material usage quantity is bigger in the research process.At present every film microslide only can use once, has two reasons to cause the not reproducible utilization of this microslide: at first be cut film microslide cause mechanical damaged; Second reason is that tissue, nucleic acid, albumen and other small-molecule substances that is bonded on the film is difficult to remove.Why difficulty is removed, and is because film does not have the such intensity of common microslide of glass material, can not be in the scraping of the surface of film; Film and solid supporting body are to be bonded together, so the film microslide can not stand High Temperature High Pressure; Film is made by special substance and is made film have special adhesion, need take into account the adhesion that can not damage film during the cleaning film surface mass; The film and the tissue specimen that have adhesion are bonding very firm, and commonsense method extremely difficulty is separated both, even needed high temperature during antigen retrieval in the SABC process (95 ℃ 10 minutes) all is difficult to sample is come off from film.
For first kind of reason,, have no idea to avoid because be damage property.But, why this method is called " micro-dissections ", be because the part that will cut is to choose the purpose sample under field of microscope (generally using 100 times, 200 times), the area of each cutting film that damages even less than 1/100, vast remaining film provides the space that utilizes again, therefore very is necessary to propose the solution at second kind of reason.
Be used to handle the reagent bottle of materials such as containing discarded sample, nucleic acid, albumen and the method for orifice plate at present and mainly contain methods such as machinery/washing agent cleaning, strong acid/highly basic soaked overnight, High Temperature High Pressure, ultraviolet ray irradiation, cobalt 60 irradiations.These measures all can not reach not only complete removal residue but also keep the purpose of sticking characteristic and integrality of film.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art part and provides a kind of tissue, nucleic acid, albumen and other small-molecule substances of consumptive material remained on surface of making to be removed, the integrality and the adhesion effect that can fully keep micro-dissections film microslide simultaneously again, carry out smoothly with the guarantee subsequent experimental, thereby the micro-dissections film microslide that the film microslide is at utmost utilized cleans the method for utilizing again.
For achieving the above object, the present invention is achieved in that
Micro-dissections film microslide cleans and utilizes method again, can implement successively as follows:
(1) the film microslide is placed boric acid, alcohol and EDTA mixed solution A soak, rock 3～5 minutes, and repeat once;
(2) the distilled water flushing is 2～3 minutes; The film microslide is transferred in the Proteinase K solution, hatched 1～2 hour for 56 ℃;
(3) microslide is placed clear water, touch sample on the film in the film microslide with the finger that has a plastic gloves, the bulk sample is removed in the distilled water flushing;
(4) microslide is placed hydrochloric acid, alcohol and EDTA mixed solution B soaked 5～10 hours; With distilled water and absolute ethyl alcohol alternate repetition flushing membrane microslide 5～10 times, and make last flushing end at absolute ethyl alcohol;
(5) the film microslide is transferred to super-clean bench rapidly, ventilates, absolute ethyl alcohol is dried up rapidly, simultaneously, open uviol lamp two surfaces of film microslide were shone respectively 30～50 minutes.
As a kind of preferred version, the pH of Proteinase K solution is 5～8 in the step of the present invention (2), and concentration is 80～150ug/ml.
As another kind of preferred version, the concentration of step of the present invention (1) mixed solution A mesoboric acid is 1%; The concentration of alcohol is 70% in described step (1) mixed solution A, and the concentration of EDTA is 0.25mol/L in described step (1) mixed solution A.
Further, the volume ratio of step of the present invention (1) mixed solution A mesoboric acid, alcohol and EDTA is 0.5～1.5: (96.5～99): 0.5～2.
Further, the concentration of hydrochloric acid is 1～1.5% among step of the present invention (4) the mixed solution B; The concentration of alcohol is 30% among described step (4) the mixed solution B; The concentration of EDTA is 0.25mol/L among described step (4) the mixed solution B.
In addition, the volume ratio of hydrochloric acid, alcohol and EDTA is 0.5～1.5 among step of the present invention (4) the mixed solution B: (96.5～99): 0.5～2.
Secondly, the present invention is preceding in step (1), and the film microslide that micro-dissections can be finished places dimethylbenzene, and 45～50 ℃ of incubators were hatched 20～30 minutes.It should be noted that the frozen tissue sample need not this step.
Compared with prior art, the present invention has following characteristics:
1, do not relate to high temperature, strong acid-base etc. in the whole operation flow process to the prejudicial factor of film;
2, by using mixed solution A, mixed solution B, guaranteed in cleaning process, the adhesion of the film that adequately protects, through the film microslide proterties of above-mentioned processing almost without any change;
3, after the ultraviolet ray irradiation, can make the stickiness of the organic membrane on the microslide reach optimum condition;
4, do not contain testing influential composition on the microslide after the cleaning;
5, required reagent is the conventional reagent of biology laboratory, and cheap, by glass section box, but batch operation;
6, recycling can be saved 3～5 times microslide quantity, saves a large amount of research fundings.
Description of drawings
Below in conjunction with the drawings and specific embodiments the present invention is further described.Protection scope of the present invention not only is confined to the statement of following content.
Whether Fig. 1 cleans up the 2% agarose gel electrophoresis figure that carries out for checking micro-dissections film microslide.
Embodiment
Clean the micro-dissections film microslide of residual cancerous lung tissue (paraffin embedding sample) with this method.
Get the film microslide that two micro-dissections that are pasted with cancerous lung tissue (paraffin embedding sample) finish, wherein a slice does not use this method to clean, and a slice uses this method to clean.
The film microslide that micro-dissections is finished places dimethylbenzene, and 50 ℃ of incubators are hatched 20 minutes (the frozen tissue sample need not this step); Microslide is placed immersion in boric acid, alcohol, the EDTA mixed solution A (mixed solution A is the mixed solution of 1% boric acid, 70% alcohol, 0.25mol/L EDTA, and its volume proportion is 1: 98: 1), rocks 4 minutes, and repeat once; Distilled water flushing 2 minutes; The film microslide is transferred to the Proteinase K solution of pH 6,80ug/ml, hatched 2 hours for 56 ℃; Microslide is placed clear water, touch sample on the film with the finger that has a plastic gloves, the bulk sample is removed in the distilled water flushing; Microslide placed in the hydrochloric acid, alcohol, EDTA mixed solution B (mixed solution B is the mixed solution of 1.5% hydrochloric acid, 30% alcohol, 0.25mol/LEDTA, and its volume proportion is 1: 98: 1) soaked 7 hours; With distilled water and absolute ethyl alcohol alternate repetition flushing membrane microslide 8 times, and guarantee that last flushing ends at absolute ethyl alcohol; Microslide is transferred to super-clean bench rapidly, ventilates, absolute ethyl alcohol is dried up rapidly, simultaneously, open uviol lamp two surfaces of film microslide were shone respectively 30 minutes.
Micro-dissections film microslide cleans and utilizes method again, can implement successively as follows:
(1) the film microslide that micro-dissections is finished places dimethylbenzene, and 45～50 ℃ of incubators were hatched 20～30 minutes;
(2) the film microslide is placed boric acid, alcohol and EDTA mixed solution A soak, rock 3 minutes, and repeat once; The concentration of described mixed solution A mesoboric acid is 1%, and the concentration of alcohol is 70%, and the concentration of EDTA is 0.25mol/L; The volume ratio of its mesoboric acid, alcohol and EDTA is 0.5: 96.5: 0.5.
(3) the distilled water flushing is 3 minutes; The film microslide is transferred in the Proteinase K solution, hatched 1 hour for 56 ℃; The pH of Proteinase K solution is 6, and concentration is 90ug/ml.
(4) microslide is placed clear water, touch sample on the film in the film microslide with the finger that has a plastic gloves, the bulk sample is removed in the distilled water flushing;
(5) microslide is placed hydrochloric acid, alcohol and EDTA mixed solution B soaked 7 hours; With distilled water and absolute ethyl alcohol alternate repetition flushing membrane microslide 7 times, and make last flushing end at absolute ethyl alcohol; The concentration of described hydrochloric acid is 1%, and the concentration of alcohol is 30%, and the concentration of EDTA is 0.25mol/L; Wherein the volume ratio of hydrochloric acid, alcohol and EDTA is 0.5: 96.5: 0.5.
(6) the film microslide is transferred to super-clean bench rapidly, ventilates, absolute ethyl alcohol is dried up rapidly, simultaneously, open uviol lamp two surfaces of film microslide were shone respectively 40 minutes.
Micro-dissections film microslide cleans and utilizes method again, can implement successively as follows:
(1) the film microslide is placed boric acid, alcohol and EDTA mixed solution A soak, rock 5 minutes, and repeat once; The concentration of described boric acid is 1%; The concentration of described alcohol is 70%, and the concentration of described EDTA is 0.25mol/L, and the volume ratio of its mesoboric acid, alcohol and EDTA is 1: 97: 1.
(2) the distilled water flushing is 3 minutes; The film microslide is transferred in the Proteinase K solution, hatched 2 hours for 56 ℃; The pH of Proteinase K solution is 7 in the described step (2), and concentration is 100ug/ml.
(3) microslide is placed clear water, touch sample on the film in the film microslide with the finger that has a plastic gloves, the bulk sample is removed in the distilled water flushing;
(4) microslide is placed hydrochloric acid, alcohol and EDTA mixed solution B soaked 9 hours; With distilled water and absolute ethyl alcohol alternate repetition flushing membrane microslide 9 times, and make last flushing end at absolute ethyl alcohol; The concentration of described hydrochloric acid is 1%; The concentration of described alcohol is 30%; The concentration of described EDTA is 0.25mol/L, and wherein the volume ratio of hydrochloric acid, alcohol and EDTA is 1.5: 98: 1.
(5) the film microslide is transferred to super-clean bench rapidly, ventilates, absolute ethyl alcohol is dried up rapidly, simultaneously, open uviol lamp two surfaces of film microslide were shone respectively 35 minutes.
Can the most micro-detected biomolecule be nucleic acid at present, in order to check cleaning performance, get the film of 0.5 * 0.5cm size respectively from the microslide that cleaned He do not clean, put into 200ul Eppendorf pipe, Xiang Guanzhong adds the 80ul distilled water, hatches 1 hour for 37 ℃.Take out 4ul liquid respectively as template, configuration 50ul standards system (dNTP:0.5ul, Taq enzyme: 0.5ul, the upstream and downstream primer: each 0.5ul, 10 * buffer:5ul, ddH20:43.5ul), carry out polymerase chain reaction,PCR (PCR), behind 40 cyclic amplifications, to get 6ul liquid respectively and put into 2% Ago-Gel and carry out electrophoresis such as Fig. 1, swimming lane 1 is the Marker sign; Swimming lane 2 is the sample after this method is handled, and does not amplify any band, and showing on the film after this method is cleaned has not had pollution of nucleic acid, and this film microslide can continue on for micro-dissections; Swimming lane 3 is undressed sample, and nucleic acid-templated existence is obviously arranged.
Be with being appreciated that, more than about specific descriptions of the present invention, only be used to the present invention is described and be not to be subject to the described technical scheme of the embodiment of the invention, those of ordinary skill in the art is to be understood that, still can make amendment or be equal to replacement the present invention, to reach identical technique effect; Use needs as long as satisfy, all within protection scope of the present invention.
Claims (7)
1, micro-dissections film microslide cleans and utilizes method again, it is characterized in that, implements successively as follows:
(1) the film microslide is placed boric acid, alcohol and EDTA mixed solution A soak, rock 3～5 minutes, and repeat once;
(2) the distilled water flushing is 2～3 minutes; The film microslide is transferred in the Proteinase K solution, hatched 1～2 hour for 56 ℃;
(3) microslide is placed clear water, touch sample on the film in the film microslide with the finger that has a plastic gloves, the bulk sample is removed in the distilled water flushing;
(4) microslide is placed hydrochloric acid, alcohol and EDTA mixed solution B soaked 5～10 hours; With distilled water and absolute ethyl alcohol alternate repetition flushing membrane microslide 5～10 times, and make last flushing end at absolute ethyl alcohol;
(5) the film microslide is transferred to super-clean bench rapidly, ventilates, absolute ethyl alcohol is dried up rapidly, simultaneously, open uviol lamp two surfaces of film microslide were shone respectively 30～50 minutes.
2, micro-dissections film microslide according to claim 1 cleans and utilizes method again, it is characterized in that: the pH of Proteinase K solution is 5～8 in the described step (2), and concentration is 80～150ug/ml.
3, micro-dissections film microslide according to claim 2 cleans and utilizes method again, and it is characterized in that: the concentration of described step (1) mixed solution A mesoboric acid is 1%; The concentration of alcohol is 70% in described step (1) mixed solution A, and the concentration of EDTA is 0.25mol/L in described step (1) mixed solution A.
4, micro-dissections film microslide according to claim 3 cleans and utilizes method again, and it is characterized in that: the volume ratio of described step (1) mixed solution A mesoboric acid, alcohol and EDTA is 0.5～1.5: (96.5～99): 0.5～2.
5, micro-dissections film microslide according to claim 4 cleans and utilizes method again, and it is characterized in that: the concentration of hydrochloric acid is 1～1.5% among described step (4) the mixed solution B; The concentration of alcohol is 30% among described step (4) the mixed solution B; The concentration of EDTA is 0.25mol/L among described step (4) the mixed solution B.
6, micro-dissections film microslide according to claim 5 cleans and utilizes method again, and it is characterized in that: the volume ratio of hydrochloric acid, alcohol and EDTA is 0.5～1.5 among described step (4) the mixed solution B: (96.5～99): 0.5～2.
7, the arbitrary described micro-dissections film microslide cleaning according to claim 1～6 utilizes method again, and it is characterized in that: preceding in step (1), the film microslide that micro-dissections is finished places dimethylbenzene, and 45～50 ℃ of incubators were hatched 20～30 minutes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100121560A CN101576653B (en) | 2009-06-22 | 2009-06-22 | Method for washing and recycling membranate glass slide for microdissection |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009100121560A CN101576653B (en) | 2009-06-22 | 2009-06-22 | Method for washing and recycling membranate glass slide for microdissection |
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| CN101576653A true CN101576653A (en) | 2009-11-11 |
| CN101576653B CN101576653B (en) | 2011-06-15 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102586107A (en) * | 2012-02-21 | 2012-07-18 | 广东温氏食品集团有限公司 | Method for recovering flaky carrier |
| CN104785486A (en) * | 2013-07-16 | 2015-07-22 | 重庆医药高等专科学校 | Cleaning method for microscopic slide touching with cedar oil stain |
| CN105085604A (en) * | 2015-08-12 | 2015-11-25 | 上海交通大学 | Recycling method for fixed probes on microarray chip |
| CN105128503A (en) * | 2015-09-07 | 2015-12-09 | 深圳市路维光电股份有限公司 | Optical film prying and removing method |
| CN108687087A (en) * | 2017-04-06 | 2018-10-23 | 希森美康株式会社 | Smear the cleaning method and smear slide preparing apparatus and smear slide preparing of component |
| CN111438087A (en) * | 2020-03-23 | 2020-07-24 | 上海市宝山区仁和医院 | Washing method for cover plate of full-automatic immunohistochemical instrument |
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2009
- 2009-06-22 CN CN2009100121560A patent/CN101576653B/en active Active
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586107A (en) * | 2012-02-21 | 2012-07-18 | 广东温氏食品集团有限公司 | Method for recovering flaky carrier |
| CN102586107B (en) * | 2012-02-21 | 2014-01-29 | 广东温氏食品集团股份有限公司 | Method for recovering flaky carrier |
| CN104785486A (en) * | 2013-07-16 | 2015-07-22 | 重庆医药高等专科学校 | Cleaning method for microscopic slide touching with cedar oil stain |
| CN104785485A (en) * | 2013-07-16 | 2015-07-22 | 重庆医药高等专科学校 | Method for cleaning histochemistry test glass slide |
| CN105085604A (en) * | 2015-08-12 | 2015-11-25 | 上海交通大学 | Recycling method for fixed probes on microarray chip |
| CN105128503A (en) * | 2015-09-07 | 2015-12-09 | 深圳市路维光电股份有限公司 | Optical film prying and removing method |
| CN105128503B (en) * | 2015-09-07 | 2019-02-01 | 深圳市路维光电股份有限公司 | Optical film sled removes method |
| CN108687087A (en) * | 2017-04-06 | 2018-10-23 | 希森美康株式会社 | Smear the cleaning method and smear slide preparing apparatus and smear slide preparing of component |
| CN108687087B (en) * | 2017-04-06 | 2022-05-13 | 希森美康株式会社 | Method for cleaning smear member and smear sample producing device |
| CN111438087A (en) * | 2020-03-23 | 2020-07-24 | 上海市宝山区仁和医院 | Washing method for cover plate of full-automatic immunohistochemical instrument |
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| CN101576653B (en) | 2011-06-15 |
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